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Dynorphin A-( L-L 7) Induces Alterations in Free Fatty Acids, Excitatory Amino Acids, and Motor Function Through an Opiate- Receptor-Mediated Mechanism

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Dynorphin A-( L-L 7) Induces Alterations in Free Fatty Acids, Excitatory Amino Acids, and Motor Function Through an Opiate- Receptor-Mediated Mechanism The Journal of Neuroscience, December 1990, IO(1‘2): 37934900 Dynorphin A-( l-l 7) Induces Alterations in Free Fatty Acids, Excitatory Amino Acids, and Motor Function Through An Opiate- Receptor-Mediated Mechanism Rohit Bakshi,’ Amy H. Newman,2 and Alan I. Faden’ ‘Center for Neural Injury, Department of Neurology, University of California, San Francisco, California 94121 and Neurology Service (127), Department of Veterans Affairs, San Francisco, California 94121, and *Department of Applied Biochemistry, Walter Reed Army Institute of Research, Washington, DC. 20307-5100 The endogenous opioid dynorphin A-( l-1 7) (Dyn A) has been Phamacological studies with opioid-receptor antagonists sup- implicated as a mediator of tissue damage after traumatic port the concept that endogenous opioids contribute to the spinal cord injury (TSCI) and causes hindlimb paralysis when pathophysiology of tissue damageafter CNS trauma (Faden et administered intrathecally. Motor impairment following in- al., 1981; Flamm et al., 1982; Hayes et al., 1983; Arias, 1985; trathecal Dyn A is attenuated by antagonists of excitatory Inoue, 1986; McIntosh et al., 1987). Dynorphin A (Dyn A), a amino acids (EAAs); whether opioid receptors mediate such 17-amino acid opioid peptide thought to be an endogenous injury has been questioned. TSCI causes various biochem- ligand for the K-Opiate receptor (Chavkin and Goldstein, 1981; ical changes associated with secondary tissue damage, in- Yoshimura et al., 1982), has been implicated as a secondary cluding alterations in tissue amino acids, phospholipids, and injury factor after spinal cord (Faden et al., 1985) and brain fatty acids. Such changes reflect injury severity and corre- trauma (McIntosh et al., 1987). Following traumatic spinal cord late with motor dysfunction. The present studies examined injury (TSCI), levels of dynorphinlike immunoreactivity in- whether dynorphin administration causes similar biochemi- creasein proportion to the degreeoftrauma (Faden et al., 1985), cal alterations and whether effects of Dyn A can be modified whereastreatment with dynorphin antiserum or K-selectiveopi- by treatment with opioid-receptor antagonists. At 24 hr after ate-receptor antagonistsimprove neurological recovery (Faden, intrathecal Dyn A, there were significant declines in tissue 1990). levels of glutamate, aspartate, and glycine. Increases in total Consistentwith its putative pathophysiologic role, Dyn A and free fatty acids were found at 2 and 24 hr, reflecting changes related fragments, administered intrathecally, causechronic pa- in both saturated and unsaturated components, which were ralysis (Faden and Jacobs, 1984; Stevens and Yaksh, 1986), loss associated with significant decreases in tissue cholesterol of the tail-flick reflex (Herman and Goldstein, 1985), neuroan- and phospholipid phosphorus at the earlier time point. Each atomical damage (Caudle and Isaac, 1987; Long et al., 1988), of these neurochemical changes, as well as corresponding and a decreasein local spinal cord blood flow (Long et al., 1987; motor deficits, were limited by pretreatment with the opioid Thornhill et al., 1989). Effects of dynorphin on the tail-flick antagonist nalmefene. In separate experiments, both nal- reflex or motor function are prevented by treatment with NMDA mefene and the selective K-opioid antagonist nor-binaltor- antagonists(Caudle and Isaac, 1988; Long et al., 1989a; Bakshi phimine (nor-BNI) limited dynorphin-induced motor dysfunc- and Faden, 1990a,b), suggestingthat dynorphin-induced neu- tion; effects of nor-BNI were dose related, and those of rological dysfunction involves the releaseof excitatory amino nalmefene were stereospecific. Therefore, behavioral and acids (EAAs). However, the role of opiate receptors has been neurochemical consequences of Dyn A administration are more controversial. Some groups have shown that opiate-re- mediated in part through opiate receptors, most likely K-e- ceptor antagonistsattenuate Dyn A-induced paralysis (Przew- ceptors. These studies indicate that phospholipid hydrolysis locki et al., 1983; Spampinato and Candeletti, 1985; Faden, and release of EAAs may contribute to dynorphin-induced 1990), whereasothers have not (Herman and Goldstein, 1985; tissue damage, suggesting for the first time a potential link- Stevensand Yaksh, 1986; Longet al., 1988,1989b). In addition, age among opioid, excitotoxin, and membrane lipid mech- nonopioid fragments of Dyn A, including Dyn A-(2-1 7) and anisms of secondary injury after neurotrauma. Dyn A-(3- 13), also causeparalysis, though with markedly less potency than Dyn A-( 1- 17) (Faden and Jacobs, 1984; Stevens and Yaksh, 1986). A recent report critically reviews this con- Received Mar. 12, 1990; revised June 21, 1990; accepted July 24, 1990. troversy and provides experimental evidence that both opioid This work was supported by NIH Grant ROl NS23422. We wish to thank and nonopioid mechanismsplay a role in Dyn A-induced pa- Florence Cheng, Brendan Chan, and Vicky Cardenas for technical assistance and Dr. Steven Graham for reviewing this work. We adhered to the principles enu- ralysis (Faden, 1990). merated in the Guide for the Care and Use of Laboratory Animals, prepared by TSCI causesincreased tissue levels of free fatty acids (FFAs; the Committee on Care and Use of Laboratory Animals of the Institute of Lab- Demediuk et al., 1985; Faden et al., 1987) and decreasedtissue oratory Resources, National Research Council [DHEW Pub. No. (NIH) 85-23, 19851. R.B. is a recipient of an Alpha Omega Alpha Research Scholarship to levels of EAAs (Demediuk et al., 1989). Releaseof FFAs after Medical Students. trauma reflects phospholipid hydrolysis and may contribute to Correspondence should be addressed to Alan I. Faden, M.D., Neurology Service (127) Department of Veterans Affairs, 4 150 Clement Street, San Francisco, CA subsequenttissue damage either through direct toxic effects(Chan 94121. et al., 1983) or through the actions of such metabolic products Copyright 0 1990 Society for Neuroscience 0270-6474/90/123793-08$03.00/O as thromboxanes (Hsu et al., 1985). Early releaseof EAAs into 3794 Bakshi et al. * Dynorphan-Induced Neurochemical Changes the extracellular space after spinal cord trauma (Panter et al., ing with a solution of 2-p-toluidinyl napthalene-6-sulfonate (Jones et 1990) is believed to lead to subsequent loss of total tissue levels al., 1982). Lipid bands were scraped from the plates prior to analysis. Cholesterol, in the presence of silica gel, was quantitated by the method (Demediuk et al., 1989). Pharmacological studies indicate that of Bowman and Wolf (1962). FFAs were extracted from the silica gel these EAA changes contribute to delayed tissue damage after using chloroform : methanol (2: 1, vol/vol). Silica gel was removed by spinal cord trauma (Faden and Simon, 1988) or brain trauma filtering through 0.2-pm nylon filters into conical centrifuge tubes. A (Hayes et al., 1988; Faden et al., 1989). The present experiments heptadecanoate internal standard was added, and the filtrate was evap- were intended to explore whether changes in FFAs and EAAs orated under N,. Fatty acid methyl esters (FAMES) were prepared using a modification of the method of Allen et al. (1984). To each centrifuge in Dyn A-induced injury parallel those after traumatic injury tube, 20 ~1 0.5 M NaOH, 80 J N,N-dimethyl acetamide, and 40 J and whether such changes are opioid-receptor mediated. methyl iodide was added, with vortexing after every addition. The re- action mixtures were then heated at 65°C for 10 min and allowed to Materials and Methods cool. Ninety microliters pyridine was added with vortexing, and the Intrathecal infusion model. Male Sprague-Dawley rats, weighing 300- tubes were again heated at 65°C for 10 min. After cooling, 0.8 ml 0.1 350 gm, were anesthetized with sodium pentobarbital(70 mg/kg, i.p.). M phosphoric acid equilibrated with ethylene chloride was added, fol- An intrathecal line was implanted to the eighth thoracic vertebral level lowed by 25 ~1 ethylene chloride. The tubes were vortexed for 30 set using a modification of the method of Yaksh and Rudy (1976), as and centrifuged at 500 x g for 2 min. Two microliters of the ethylene previously detailed (Bakshi and Faden, 1990a). Briefly, polyethylene chloride lower phase was removed with a Hamilton syringe and injected tubing (PE- 10) was implanted into the subarachnoid space through the into a Perkin-Elmer Sigma 300 gas chromatograph for separation and atlanto-occipital membrane and passed to T8. The catheter was then auantitation of FAMES. A Sunelco (Bellefonte. PA) nrenacked 5% DEGS- secured below the skin and the wound sutured. Animals were allowed PS (Supelcoport 100/200 mesh) column was ‘used wi<h N, as the carrier 24 hr to recover, at which time those showing motor deficits were gas for all gas-liquid chromatography experiments. The initial column removed from the experiment. Agents were infused through the in- temperature was 180°C and was linearly increased at a rate of 4”C/min. trathecal catheter in a vehicle of 20 ~1 physiologic saline. The injector and flame-ionization detector temperatures were set at Study 1. Animals were randomly assigned to pretreatment with either 250°C. Detector linearity was checked with known standards. Chro- nalmefene (8 nmol; Key Pharmaceuticals, Miami, FL) or vehicle in a matograms were recorded, and peak areas were calculated using the volume of 10 ~1. Fifteen minutes later, Dyn A-(1-1 7) (24 nmol; Pen- chromatographic data station listed above. insula Laboratories,
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